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12. TRANSDUCTION MECHANISMS G

Katerina J. Damjanoska and Louis D. Van de Kar∗

1. INTRODUCTION

Receptors can be classified into three large classes: gated ion channels; genotropic receptors, which can act as factors; and G -coupled receptors (GPCRs). GPCRs are membrane proteins with a unique seven- transmembrane transversing structure (hepta-helix). GTP-binding proteins (G proteins) transmit extracellular signals from -surface receptors to intracellular effectors such as , adenylyl cyclases, and ion channels. G proteins are a family of trimeric proteins, consisting of α, β, and γ subunits. The α subunits of G proteins bind guanine nucleotides (GTP and GDP) with high affinity and specificity (Dessauer 1996; Neer 1995). It is this affinity for guanine nucleotides that gives them their name G proteins. Approximately 1% of the mammalian genome encodes for G-protein coupled receptors (Morris 1999), and approximately 50% of pharmaceuticals target receptors, largely GPCRs (Drews 2000). Although no drugs have been developed, so far, that specifically act on G proteins, G proteins are potential pharmaceutical targets as changes in G proteins and their associated regulatory proteins have been implicated in a number of pathological conditions. Since the discovery of G proteins in the late 60’s and early 70’s of the 20th century, by Alfred G. Gilman and , research dedicated to the study of G proteins has increased dramatically. G proteins associate with a variety of receptors (See Table 1). This enables G proteins to be intracellular transducers of a variety of extracellular signals such as , , odorants, and

∗ Katerina J. Damjanoska and Louis D. Van de Kar, Ph.D., Center for Serotonin Disorders Research and Department of Pharmacology & Experimental Therapeutics, Loyola University Chicago, Stritch School of Medicine, Maywood, Illinois, 60153 U.S.A.

289 290 K.J. DAMJANOSKA ET AL. photons. This chapter will provide an overview of the variety of subtypes and their associated proteins. As this review will focus on psychiatric disorders, we will limit our discussion to the importance of G proteins in the central nervous system.

2. TYPES OF HETEROTRIMERIC G PROTEINS

Heterotrimeric G proteins (Gαβγγ) are currently categorized according to the Gα subunit, historically thought to be the only active subunit of the G protein trimer. As there are four main types (classes or families) of Gα subunits, there are four main classes of heterotrimeric G proteins. The classes of Gα proteins, and hence of G protein trimers, are Gαs, Gαq, Gαi, and Gα12. Each class of Gα proteins has subtypes (See Section 2.2). Although multiple subtypes of Gα proteins have been identified, there are not as many Gα proteins as there are G protein-coupled receptors (GPCRs). Table 1 shows the different classes of Gα proteins and some of the receptors to which they are coupled. Many GPCRs couple to the same Gα protein subtype, yet are still capable of mediating their specific cellular and physiologic effects. This overlap in proteins can be partially explained by differential expression of proteins in cells. Yet, there are numerous cells that express more than one GPCR and multiple subtypes of Gα proteins. One theory proposes that cellular microdomains with -rich regions (lipid rafts) in the preferentially aggregate the required and relevant proteins within close proximity of their respective receptors. Gα proteins and their effector can be localized to microdomains by their association with specific proteins of the membrane and , such as (See Section 5) (Donati 2003; Huang 1997). Gα proteins can also undergo a variety of lipid modifications that may assist in targeting Gα proteins to subcellular compartments, caveolae, and lipid microdomains (See Section 5). Furthermore, not all Gα protein subtypes are 7 sequestered to the same microdomain. For instance, Gαq proteins are normally 7 found in caveolae without being associated to Gβγγ proteins. On the other hand, while

Gαi and Gαs proteins can be found in caveolae, they are predominantly found in lipid rafts complexed to Gβγγ proteins (Oh 2001). The theory of cellular compartmentalization of the Gα proteins provides a plausible explanation for the speed of selection (efficiency) and selectivity of -to-G protein signaling. Another emerging theory concerning receptor-to-G protein coupling is “agonist- directed trafficking of receptor stimulus” (Kenakin 1995). This theory suggests that ligands can induce different conformational changes in the receptor so that one receptor can activate multiple Gα protein-mediated signaling cascades (Kenakin 1995 ; Berg 1998). G PROTEINS 291

Table 1. Gα protein families and the receptors that are coupled to them. Some of the information reported in this table for receptor-G protein coupling is obtained from in vitro reconstitution or studies in cell culture and are not confirmed in vivo. The results obtained from reconstitution or cell culture studies must be taken with caution as the protein levels and ratios of purified or transfected receptors and G proteins may exceed the physiological levels of the proteins and result in otherwise unlikely interactions. These in vitro studies may also lack the associated proteins critical for the association between receptors and G proteins. Gα Protein Associated Receptors References Family

Gαs Adenosine (A2A, A2B) 10, 11, 12, 13

Adrenergic [α2A, α2B, α2C (formerly α2C10, α2C2, α2C4), β1, β2, β3] 14, 15, 16, 17 (CTR) 18 Complement (C5A) 19 Corticotropin-releasing (CRH-R1, -R1α, -R2) 20, 21 (D1, D3, D5) 22 (ETAR) 23, 24 (GR) 25 -releasing hormone (GnRH-R) 26 Histamine (H2) 27 /chorionic gonadotropin (LH/CG R) 28 Melanocortin (MC1R, ACTHR, MC3R, MC4R, MC5R) 29 (PTH) 30, 31 (IP, EP2, EP4, DP, EP3B, EP3C) 32, 33 Serotonin (5-HT4, 5-HT6, 5-HT7) 34, 35, 36 (SPR or NK1R) 37, 38 Stimulating Hormone (TSH) 39 (V2) 40 Gαi Adenosine (A1 and A3) 41, 42, 43 Adrenergic (α2A, α2B, α2C) 44, 17 Angiotensin (AT1) 45 Bombesin 46 (B1, B2) 47, 48, 49, 50 Calcitonin (CTR) 51, 52 (CB1, CB2) 53, 54 Cholecystokinin (CCKB) 55 292 K.J. DAMJANOSKA ET AL.

Table 1. (continued) Gα Protein Associated Receptors References Family Gαi Compliment (C3A, C5A) 56, 57, 58 Corticotropin-releasing hormone (CRH-R1α) 20 Dopamine (D1, D2S, D2L, D3, D4) 22 Endothelin (ETBR) 23, 24 Galanin (GALR1, GALR2) 59, 60 Glutamate (mGluR2, mGluR4) 61, 62 Gonadotropin-releasing hormone (GnRH-R) 26 Histamine (H3) 63 -like (IGF IR) 64 Luteinizing hormone/chorionic gonadotropin (LH/CG-R) 28 Lysophosphatidic Acid (LPA) 65 Melatonin 66, 67 Muscarinic acetylcholine (m2 and m4) 68, 69 Y* (Y1, Y2, Y4, Y5) 70, 71 Neurotensin* (NTS1) 72, 73, 74 75, 76, 77, Opioid (*µ, *κ and *δ) 78, 79 Orphanin/Nociceptin (OFQR) 76 Oxytocin (OTR) 80, 81 Parathyroid hormone (PTH) 30 Platelet-activating factor (PAF) 82 Prostaglandin E (EP3A, EP3D, CRTH2) 83, 32, 33 Serotonin (*5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, 5-HT1F, 5- 34, 84, 85, HT5A) 35, 36 (SRIF) 86, 87 Substance P (SPR or NK1R) 37, 88 Thrombin 89 Thyroid Stimulating Hormone (TSH) 90 Gαz Compliment (C5a) 91 Dopamine (D2S, D2L, D3, D4, D5) 22 Formyl peptide (fMLP) 91, 92 Melatonin 66 93, 94, 95, Opioid (*µ, κ and δ) 77, 96, 78 G PROTEINS 293

Table 1. (continued) Gα Protein Associated Receptors References Family Gαz Serotonin (*5-HT1A) 97 Gαq Adenosine (A2A, A2B, A3) 98, 99, 41 Adrenergic (α1, α2A) 100, 101 Angiotensin (AT1) 102 Bombesin (GRP-R, NMB-R, BRS-3) 46, 103, 104 47, 48, 49, Bradykinin (B1, B2) 50 Calcitonin (CTR) 105 Cholecystokinin (CCKA, now CCK2; CCKB, now CCK1) 106, 55, 107 Compliment (C5A) 108 Corticotropin-releasing hormone (CRH-R1α) 20 Dopamine (D3) 109 Endothelin (ETAR, ETBR) 24 Galanin (GALR2) 59 Glutamate (mGluR1, mGluR5) 110, 111 Gonadotropin-releasing hormone (GnRH-R) 112, 113 Gαq Histamine (H1, H2) 27 (continued) Lysophosphatidic Acid (LPA) 114 Melanocortin (MC3R) 115 Muscarinic (m1, m5) 68, 116 Neurokinin (NK2) 117, 118 Neurotensin (NTS1) 73 Orexin (types 1) 119 Oxytocin 80 Platelet-activating factor (PAF) 82 Prostaglandin (TP, IP, FP, EP3D) 32, 33 Purinoceptor (P2Y) 120 Serotonin (5-HT2A, 5-HT2B, 5-HT2C) 34, 35, 36 Substance P (NK1R or SPR) 37, 121 Thrombin 122 Thyroid Stimulating Hormone (TSH) 39 Vasopressin (V1a, V1b) 104, 123 294 K.J. DAMJANOSKA ET AL.

Table 1. (continued)

Gα Associated Receptors References Gα12 Adrenergic (α1) 124 Bombesin (GRP-R) 46 Endothelin (ETBR) 125 Galanin (GALR2) 59 Lysophosphatidic Acid (LPA) 126 Prostaglandin (TP) 32 127 Thyroid Stimulating Hormone (TSH) 90 * Denotes receptor/G protein interactions confirmed in vivo. Studies performed in vivo consisted of 1) receptor and Gα protein colocalization depicted by immunohistochemistry, 2) identification of the Gα protein family mediating the specific response by treatment with or toxin, or 3) in vivo suppression of expression of specific Gα proteins by antisense oligodeoxynucleotides. Abbreviations: CRTH2, chemoattractant receptor-homologous molecule expressed on T-helperpyp type 2 cells; EP, prostag- landin E (PGE22 ) receptor; IP, prostaglandin I (PGI22 ) receptor; DP, prostaglandin D (PGD 2 ) recep- tor; FP, prostaglandin F (PGF2α) receptor; TP, (TXA2) receptor.

2.1.Regulation of G protein signaling

Receptors are coupled to the trimeric form (αβγγ) of G proteins. When a receptor agonist binds to its receptor it induces a that increases the 2+ 2+ affinity of the Gα protein for Mg . Once Mg is bound to the Gα protein, it stimulates the release of guanine diphosphate (GDP) from the Gα protein, and the binding of guanine triphosphate (GTP) to the Gα protein, promoting the dissociation of the Gβγγ from the Gα protein. The GTP-bound Gα protein and the Gβγ protein dimer are then capable of stimulating their respective effector enzymes. Hydrolysis of an inorganic phosphate from Gα-GTP converts it to Gα-GDP, decreasing its high affinity for second messenger enzymes and increasing its affinity for the Gβγγ protein dimer. The result is re-association of the Gα and Gβγγ proteins and the formation of the Gαβγγ protein trimer (Fig. 1). The GDP-bound Gαβγγ protein trimer can then couple to its receptors. While this scheme represents the current kinetic model of G protein signaling, the exact biochemical and structural dynamics among the Gαβγγ trimer, the receptor, and the effector proteins are still under investigation (Dessauer 1996). The activity of the G protein subunits can be regulated by accessory proteins and by post-translational modifications. Post-translational regulation of G protein subunits will be discussed in Section 5. Accessory proteins regulate the activity of Gα proteins, Gβγγ proteins, and their association with each other. Two examples of accessory proteins are the regulators of G protein signaling (RGS) proteins and G PROTEINS 295 phosducin. Gα proteins have an intrinsic GTPase activity that in many cases is very slow. This intrinsic GTPase activity of Gα proteins is enhanced by RGS proteins or by some effector enzymes, such as Cβ. RGS proteins bind directly to Gα proteins, with some apparent subtype specificity, and accelerate the hydrolysis of GTP and thereby catalyze the inactivation of the Gα proteins (Fig. 1) (Berman 1998 ; Berghuis 1996 ; Berman 1996). A second method of regulating the activity of G proteins is to prevent the formation of the Gαβγγ protein trimer by inhibiting the Gβγγ protein subunits from associating with the Gα proteins (Fig.1). Phosducin is a 33-kDa protein that has been characterized in the and pineal gland (Reig 1990; Lee 1987) but is also expressed in various other tissues, such as the brain, liver, lung, and heart (Danner 1996). Phosducin inhibits Gβγγ proteins from reassociating with GDP-bound Gα proteins and induces translocation of the Gβγγ proteins to the cytoplasm (Tanaka 1996). This translocation of Gβγγ proteins to the cytoplasm is thought to be due to phosducin binding to the region on the Gβγγ protein dimer that is implicated in membrane binding and receptor interaction (Tanaka 1996; Lambright 1996; Sondek 1996). of phosducin by protein A (PKA) at residue 73 reduces the stability of the interaction between phosducin and the Gβγγ protein dimer, resulting in the reassociation of the Gβγγ protein dimer with the Gα protein (Gaudet 1999; Yshida 1994). In vitro studies have shown phosducin to inhibit β-adrenoceptor-stimulated activity (Danner 1996). The ability of phosducin to inhibit receptor-mediated adenylyl cyclase activity can be attributed to phosducin sequestering the Gβγγ protein dimers and thereby decreasing the available pool of intracellular G protein trimers. Additional studies have shown that: 1) phosducin (IC50 = 100 µM) decreases Gβγγ protein-mediated activation of adenylyl cyclase and -β (PLCβ) and 2) phosducin (IC50 = 15 nM) decreases the GTPase activity of Gαo proteins in the presence of Gβγγ protein dimers, but not of 140 isolated Gαo proteins. These in vitro studies not only demonstrate that phosducin is capable of disrupting the Gβγγ-effector interaction, but that phosducin is approximately 5 fold more effective in disrupting the Gβγγ-Gα protein interaction (Bluml 1997). Phosducin is potentially a useful Gβγ protein inhibitor. While our knowledge about phosducin is still in its infancy, it presents a possible therapeutic approach that can reduce a supersensitized signal without targeting the receptor protein. 296 K.J. DAMJANOSKA ET AL.

Figure 1. Regulation of G protein signaling. Abbreviations: A, agonist; GDP, ; 2+ GTP, ; Pdc, phosducin; Pi, inorganic phosphate; Mg , magnesium ion; RGS, regulator of G protein signaling.

2.2. Gα protein subunits

Approximately 20% of the primary sequence of Gα subunits is composed of conserved amino acids. Gα protein subunits are categorized into 4 classes: Gαs, Gαi, Gαq, and Gα12. These 4 classes or families of Gα protein subunits are based on sequence similarities aside from the conserved amino acids present in all Gα protein subunits (Hepler 1992; Wilkie 1992). Each G protein family is named based on the prototypical Gα protein family member. Most of the current research focuses on the function of Gα proteins at the intracellular portion of the cell membrane and its role in GPCR signaling. Although Gα proteins have been localized in the Golgi complex (Nagahama 2002; Ercolani 1990), endoplasmic reticulum (Audigier 1988), and endosomal structures (Colombo 1992) the function of Gα proteins in these subcellular compartments is not entirely understood. It is possible that localization to these subcellular regions may enable Gα protein subunits to influence the formation and transport of secretory vesicles (Leyte 1992; Stow 1991; Tooze 1990), affect intra-compartmental protein transport (Schwaninger 1992), and regulate Golgi structure (Nagahama 2002). G PROTEINS 297

The Gαs protein family consists of Gαs and Gαolff proteins, with long (Gαs-L) and short (Gαs-S) splice variants of the Gαs protein (Kozasa 1988; Robishaw 1986). Both proteins activate adenylyl cyclase and are adenosine 5’-diphosphate (ADP)- ribosylated by (Vibrio cholera) on an arginine residue (Gilman 1989). This covalent, post-translational modification inhibits the intrinsic GTPase activity of the Gαs protein family, which hinders the inactivation of the proteins in Gαs family. Gαs proteins are widely distributed in various tissues, including the brain (Dessauer 1996). Gαolff proteins are associated only with the olfactory areas of the brain. In the gastrointestinal system, defects in the termination of Gαs protein activity are attributed to the etiology of the diarrhea induced by Vibrio cholerae. Other pathologies associated with defective termination of Gαs protein activity include acromegaly, McCune-Albright syndrome, and some thyroid adenomas (Farfel 1999).

Pseudohypoparathyroidism type I is caused by an inactivation or genetic loss of Gαs proteins.

The Gαi protein family consists of Gαi-1, Gαi-2, Gαi-3, Gαz, Gαo-1, Gαo-2, and Gαt ( specific) proteins (Dessauer 1996). The Gαi protein family inhibits adenylyl + cyclase activity and also opens K ion channels. The retina-specific Gαt protein is the only member that activates cGMP phosphodiesterase. All the members of the Gαi protein family, except for Gαz proteins, are ADP-ribosylated by pertussis toxin (Bortadella pertusis) on a conserved COOH-terminal cysteine residue (Ui 1990).

After this covalent modification occurs, the proteins of the Gαi family are uncoupled from their receptors and their intracellular signaling is inactivated. Many investigators have used pertussis toxin in order to determine whether proteins of the

Gαi family play a role in specific receptor-mediated intracellular signaling cascades. All the members of Gαi protein family are expressed in a variety of tissues, including the brain. Defects in the termination of Gαi protein activity are implicated in the etiology of some adenomas of the and endocrine tumors of the ovary

(Farfel 1999). ADP-ribosylation of Gαi proteins by hinders the activation of Gαi proteins and causes whooping cough. A point mutation genetically inactivates Gαt proteins in affected individuals of dominantly inherited congenital stationary night blindness (Dryja 1996).

The Gαq family of proteins consists of Gαq, Gα11, Gα14, Gα15, and Gα16 proteins. Phospholipase C (PLC) is activated by all members of the Gαq protein family. Proteins of the Gαq family do not undergo ADP-ribosylation by either cholera toxin or pertussis toxin. Gα11 and Gαq proteins are widely distributed in various tissues, 155 including the brain. Gα15 and Gα16 proteins are specifically found in myeloid and lymphoid tissue while Gα14 proteins are found in a variety of tissues but not in brain (Dessauer 1996). While none of the proteins of the Gαq family have been associated with the etiology of a disease, a change in levels of Gαq/11 proteins was observed in brain areas affected by Alzheimer’s disease158 and by antidepressant treatment (Lesch 1992). 298 K.J. DAMJANOSKA ET AL.

Finally, the family of Gα12 proteins consists of Gα12 and Gα13 proteins. Both are widely distributed in various tissues, including the brain (Dessauer 1996). Gα12 and + - Gα13 proteins were suggested to regulate the activity of Na /Cl antiporters (Offermanns 1996), activate small G proteins (Rho) (Mao 1998), and activate mitogen-activated protein , such as c-Jun kinases (Jho 1997; Collins 1996;

Voyno-Yasenetskaya 1996; Prasad 1995). While the Gα12 protein family has not yet been implicated in any disorders, the Gα12/13 are considered to be proto- oncogenes. Out of all four G protein families, activated mutant forms of Gα12 and Gα13 proteins had the highest transformation efficiency in NIH3T3 cells (Offermanns 1994). Furthermore, a screen of human tumor cell lines has shown an increased expression of Gα12 and Gα13 proteins in a number of breast, colon, and prostate adeno-carcinoma-derived cell lines (Xu 1993).

2.3. Gβγ protein subunits

168, 169 Seven isoforms of Gβ proteins (Gβ1, Gβ2, Gβ3, Gβ3S, Gβ4, Gβ5, Gβ5L) and 11 isoforms of Gγ proteins (Gγ1, Gγ2, Gγ3, Gγ4, Gγ5, Gγ7, Gγ8, Gγ10, Gγ11, Gγ12, GγC) have been documented (Downes 1999; Gautam 1998). Two of the isoforms of the Gβ proteins are splice variants: Gβ3S protein (short form of Gβ3 protein) and Gβ5L protein (long form of Gβ5 protein). All the members of the Gβ protein family, except for the Gβ5 and Gβ5L proteins, have a great degree of homology in their amino acid sequence (Gautam 1998). The Gγ protein family has more diversity in their primary sequence than the Gβ protein family. This allows the Gγ protein subtypes to be grouped into 4 subfamilies based on sequence similarity. Group I includes Gγ1, GγC, Gγ11; Group II includes Gγ2, Gγ3, Gγ4; Group III includes Gγ7, Gγ12; and Group IV includes Gγ5, Gγ8, Gγ10. Group I Gγ proteins are predominantly expressed in the retina and Group II Gγ proteins are abundant in the nervous system. Gβ and Gγ proteins form a tight association with each other and are usually found as a dimer. With 7 isoforms of Gβ protein, 11 isoforms of Gγ protein, and over a dozen isoforms of Gα protein, there are numerous combinations of G protein trimers that can be formed in the cell. The issue of which Gα, Gβ, and Gγ isoforms associate with one another with high selectivity has been addressed using transfected cell assay systems (Zhou 2000; Pronin 1992), yeast two-hybrid systems (Yan 1996), and immunoprecipitation of proteins from tissue preparations (Asano 1999). This selectivity along with differential distribution of the subunits in tissues (Brunk 1999; Betty 1998) is one possible mechanism by which so many receptors use the same G protein subunits to elicit a variety of specific cellular and physiological responses. Gβγγ protein dimers can regulate effector enzymes and ion channels. Their importance in signal transduction was initially unknown but is now coming to the forefront of research. Gβγγ subunits are capable of regulating activity, such as activation of phosholipase A2 (PLA2) (Jelsema 1987), phospholipase C (PLC) G PROTEINS 299

(Rebecchi 2000 ; Barr 2000), and phospho-inositide (PI) 3-kinase (Stephens 1997); inhibition of adenylyl cyclase type I, activation of adenylyl cyclase type II and IV; (Taussig 1993; Tang 1991 and 1992) activation of K+ channels (Clapham 1989); and 2+ inhibition of Ca channels by Gβ1 and Gβ3 proteins (Shekter 1997). Although some patients with hypertension have been shown to contain a gain of function mutation in their Gβ3 (Siffert 1998), there is still very little information regarding disorders associated with a change in the function of Gβ or Gγ proteins.

3. RGS PROTEINS

Regulators of G protein signaling (RGS) proteins are GTPase activating proteins (GAPs) that enhance the intrinsic GTPase activity of G proteins (Hollinger 2002), but RGS proteins can regulate G protein activity by mechanisms other than their GAP activity. RGS proteins can also bind to actived Gα proteins to prevent effector activation, independent of their GAP activity (Carman 1999; Hepler 1997). In addition, the GoLoco motif, found in RGS12 and RGS14 proteins, allows RGS proteins to bind to Gαi-GDP proteins (Kimple 2001 and 2002). The ability of certain RGS proteins to bind to inactived Gα proteins provides them with guanine nucleotide (GDP) dissociation inhibitor (GDI) activity in addition to their GAP activity. Phosphorylation of RGS14 increases its GDI activity without affecting its GAP activity (Hollinger 2003). Hence, RGS proteins can catalyze the inactivation of Gα proteins, prolong the period of inactivation of Gα proteins, and also prevent Gα proteins from activating downstream effectors. Furthermore, RGS proteins can inhibit effector activation mediated by Gβγγ proteins. For instance, RGS3 inhibits Gβγ- mediated phosphate production and MAPK activation (Shi 2001). Alterations in RGS proteins have been implicated in anxiety, aggression, and schizophrenia (Mirnics 2001 ; Oliveira-dos-Santos 2000).

Table 2. RGS proteins and their associated Gα protein families. RGS Family RGS Protein Associated Gα Family References

R4 RGS1 Gαi 205 † RGS2 Gαq, Gαi 206, 207 † RGS3 Gαi, Gαq 208 † RGS4 Gαq, Gαi 188, 130, 209 † RGS5 Gαi 210, 211, 212 † RGS8 Gαi 213, 214

RGS13 Gαi, Gαq 201 † RGS16 Gαi 215, 211

RGS18 Gαi, Gαq 216, 217 † ‡ R7 RGS6 Gαo 218, 212 † RGS7 Gαi, Gαq 213, 219, 220, 212 300 K.J. DAMJANOSKA ET AL.

† ‡ RGS9 Gαt , Gαi 221, 219, 222 † ‡ RGS11 Gαo 223 † R12 RGS10 Gαi 224 † RGS12 Gαi, Gα12 161, 225 † RGS14 Gα12, Gαi 191, 189, 226 RA Axin † ND 227 Conductin † ND 228 † RL P115RhoGEF Gα12 229, 230, 161 RhoGEF ND 231 LscRhoGEF ND 232 D-AKAP2 ND 233

GRK2 Gαq 234 ‡ RGSPX1 Gαs 198 † ‡ RZ RGSZ1 Gαz 235, 236 ‡ RGSZ2 Gαz 143 ‡ RET-RGS1 Gαt 237 † GAIP Gαi, Gαq 188, 238, 130 ? RGS15 † ND 212 † Denotes RGS proteins that can undergo alternative splicing239. ‡ Denotes the specific Gα protein of a Gα protein family with which the RGS protein interacts. Most RGS proteins associate with multiple members of a Gα protein family, but there are some RGS proteins that act as GAPs for only certain members of a Gα protein family. Abbreviations: D- AKAP, dual specificity A kinase anchoring protein; GAIP, Gα interacting protein; GRK, G protein receptor kinase; ND, associated Gα protein is not yet determined; ? = RGS family not known.

RGS proteins are widely distributed in the brain. Although RGS proteins are considered soluble hydrophobic proteins, they are found in the cytosol and are also associated with the cellular membrane (Bernstein 2000; Srinivasa 1998). RGS protein subtypes are categorized into 6 families (R4, R7, R12, RZ, RL and RA) based on their domain or sequence similarities (Table 2) (Hollinger 2002; Ross 2000; Zheng 1999). All RGS proteins have GAP activity except for axin and conductin (also known as axin-like or axil), which are in the RA family of RGS proteins. The RA family is included in the classification of RGS proteins because of their sequence similarity to RGS proteins, even though they have not been shown to have GAP activity (Gold 1997). Both axin and conductin are scaffold proteins in the Wingless-type (Wnt) protein receptor pathway. The is present in various species, from invertebrates to mammals, and is important in development, cellular proliferation, and cellular differention. Most G proteins have multiple RGS proteins that can enhance their GTPase activity (Table 2). Gαs proteins are the exception. Except for RGSPX1, no other wild-type RGS proteins have GAP activity for Gαs proteins (Zheng 1999). RGS16 and RGS4 proteins can act as GAPs for Gαs proteins only after a point mutation G PROTEINS 301

(A229S) is incorporated into the Gαs protein (Natochin 1998). Yet, by attenuating the activation of the effector enzyme other RGS proteins, such as RGS2 and RGS13, can regulate Gαs activity indirectly (Kehrl 2002; Johnson 2002; Sinnarajah 2001. There is a greater variety of RGS proteins than there are Gα proteins. As depicted in Table 2, the GTPase activity of many G proteins is regulated by multiple RGS proteins. The presence of this natural redundancy may be to ensure proper control of intracellular signaling under a variety of cellular or physiological conditions. On the other hand, cell- or region-specific expression of subtypes of RGS proteins and Gα proteins may limit the possible interactions and may be one way that specificity is achieved between RGS proteins and Gα proteins (Gold 1997).

4. TRANSCRIPTIONAL REGULATION OF RECEPTOR SIGNALING

Transcriptional regulation usually signifies a change in the amount of protein produced by the cell’s transcriptional machinery. This is typically measured as a change in the level of mRNA and/or a change in the level of protein. A change in the levels of mRNA does not indisputably result in a change in the levels of protein as other post-transcriptional factors, such as protein degradation and stability of the mRNA must be considered. Both transcriptional and post-transcriptional regulation will be discussed in this section.

4.1. Transcriptional regulation of receptor signaling

Transcriptional regulation of receptor signaling can encompass a downregulation of receptors, G proteins, RGS proteins, or second messenger enzymes and other effectors. Downregulation is defined as a decrease in the amount of protein expressed or a decrease in the density of receptors labeled with an antagonist. A reduction in the density of membrane receptors, while all other signaling components remain unchanged, will decrease signal transduction across the membrane. Hence, in a dose-response experiment, the maximal response (Emax) will be reduced. An increase in the density of membrane receptors may cause a larger cellular and physiologic response if the expressed receptors are functionally coupled to their intracellular signaling components (increased Emax). A reduction in the amount of G proteins will hinder the external signal from being fully amplified between the receptor and . In a dose-response experiment, this will result in an increase of the dose of drug needed to produce 50% of the maximal response (ED50). A surplus of G proteins may allow for a more efficient transfer of the extracellular signal (increased ED50). While many treatments have been shown to alter receptor expression, chronic treatment with antidepressant drugs (clorgyline, desipramine, fluoxetine, imipramine) also produces treatment- and 302 K.J. DAMJANOSKA ET AL.

region-specific changes in protein levels and in mRNA levels of Gαs, Gαi, and Gαq proteins in rat brain (Lesch 1992). This could be one mechanism of action of certain antidepressant agents. Therefore, either changes in levels of receptor proteins or changes in levels of Gα proteins or both are possible mechanisms to alter signal transduction. A reduction in RGS proteins would lead to a decrease in the velocity of the GTPase activity of the Gα proteins. The G protein would activate second messenger enzymes for a longer time and thus would evoke a greater physiologic response (increased Emax). On the other hand, an increase in the amount of RGS protein would enhance the GTPase activity of Gα proteins and lead to a smaller physiologic response (decreased Emax). A decrease in the levels of RGS4 proteins was identified in prefrontal cortex of schizophrenic patients (Mirnics 2001). It is not known whether this change in levels of RGS4 proteins is due to a genetic factor or due to a specific adaptation to the disease. This decrease in RGS4 proteins may allow for a greater cellular response and, subsequently, greater neuronal signaling in the prefrontal cortex. The alteration in levels of RGS4 proteins may provide insight in the regional/cellular etiology of schizophrenia.

4.2. Post-transcriptional regulation of receptor signaling

Post-transcriptional regulation of signaling can occur at the level of degredation of proteins or post-translational modifications (See Section 5) that change a protein’s ability to function. Increased degredation without a compensatory increase in transcription and translation of a specific protein will eventually lead to a decrease in the total amount of the protein within the cell or in the cellular membrane. The opposite is also likely. An increase in transcription/translation without a compensatory increase of degradation will lead to an increase in total levels of the protein. While this type of regulation can occur for receptors, G proteins, RGS proteins, and effector proteins, there are not many examples of a change in the half- lives of proteins changing due to a disorder or its treatment. The agonist-induced decrease in serotonin 2A (5-HT2A) receptor density is preceeded by a decrease in 5- HT2A receptor mRNA (Anji 2001). It is possible that protein degredation may only be an immediate response to decreased protein expression before a more stable change in mRNA can occur.

5. POST-TRANSLATIONAL REGULATION OF SIGNALING

Post-translational modifications of proteins by an attachment of a phosphate side-chain (phosphorylation) or the addition of a side-chain can be integral to the proper function of the protein and its cellular localization. Phosphorylation of G proteins can change the interaction between G proteins and other proteins, such as G PROTEINS 303 receptors and RGS proteins, or change the activity of the G protein. A common example of phosphorylation changing protein function is kinase-mediated internalization of receptors and desensitization of receptor signaling (Lohse 1992). This section will focus on phosphorylation and the most commonly described lipid modifications (, and ) of G proteins and their associated proteins. Palmitoylation of Gα proteins and prenylation of Gγ proteins are important lipid modifications that assist in the association of the G protein subunits with the receptor and the bilayer lipid membrane (Fig. 2). No lipid modifications have been identified for Gβ proteins, although this is not to say that none exist. The exact mechanism as to how lipid modification targets the G protein to the bilayer membrane is unknown. The simplest explanation is that the lipid inserts directly into the hydrophobic bilayer membrane and thereby anchors the G protein to the cell membrane. In vitro assays measuring the affinity of various acylated peptides for lipid membranes showed that myristoylation and farnesylation do not have sufficient energy to dock proteins to the membrane on their own (Silvius 1994; Peitzsch 1993). Only palmitate and geranylgeranyl isoprenoids have sufficient energy to stably anchor a protein to a lipid membrane. Thus, while some lipid modifications of proteins are sufficient by themselves to target some proteins to the cell membrane, other proteins require additional factors, such as protein-protein interactions. Some lipid modifications may target the G protein to an integral membrane protein rather than the membrane. Protein-protein interactions can assist in recruiting the G protein or RGS protein to the membrane by association with membrane bound proteins (such as phosphodiesterase MIR16) (Zheng 2000), intracellular scaffolding proteins (14-3-3 proteins) (Benzing 2000), cytoskeletal proteins (tubulin) (Donati 2003), coatomer proteins on secretory membranes (β-COP) (Sullivan 2000), and binding partners such as GIPC (GAIP-interacting protein, C terminus) (Booth 2002; Lou 2001; De Vries 1998). Axin and conductin, RGS proteins of the RA family, do not have GAP activity but can serve as a scaffolding proteins for G proteins by linking the G protein to its downstream effectors (Hollinger 2002). The Gβγγ proteins are also thought to assist in recruiting the Gα protein to the cell membrane by acting as membrane-bound docking proteins. This was demonstrated by coexpressed Gβγγ and Gα proteins increasing the amount of functional, membrane-bound Gα protein (Linder 1993). Thus, some lipid modifications of Gα proteins do not directly anchor the Gα protein to the cell membrane but may anchor Gα proteins to Gβγγ proteins or other membrane-bound proteins.

5.1. Prenylation

Prenylation is the attachment of a 15-carbon farnesyl or 20-carbon geranylgeranyl isoprenoid to cysteine residues of proteins. All Gγ proteins are geranylgeranylated on their C-terminal, except for the Gγ protein subunits (Gγ1 and 304 K.J. DAMJANOSKA ET AL.

Gγ11) of the retinal trimeric G protein, which are farnesylated (Gautam 1998; Yamane 1990; Mumby 1990; Fukada 1990). The exact reason for the different type of prenylation for the retinal Gγ protein is unkown. Generally, prenylation of the Gγ protein is necessary for its localization to the membrane (Muntz 1992; Simonds 1991) but is not necessary for the formation of the Gβγγ dimer (Iniguez-Lluhi 1992). Even though the Gβγγ dimer can be formed with an unprenylated Gγ protein, the Gβγ dimer does not have high affinity interactions with Gα proteins or adenylyl cyclase (Fig. 2). This suggests that prenylation of Gγ proteins is important to cellular signaling by affecting both cellular localization and protein-protein interactions.

Figure 2. Importance of palmitoylation of Gα protein and prenylation of Gγ protein to the efficiency of signaling. Abbreviations: A, agonist; GDP, guanosine diphosphate; GTP, guanosine triphosphate; Pi, inorganic phosphate; Mg2+, magnesium ion. G PROTEINS 305

5.2. Myristoylation

Myristoylation is the attachment of a 14-carbon myristate fatty acid to a N-terminal glycine of Gα proteins. The attachment of this lipid was once thought to be critical for membrane anchoring of Gα proteins, such as Gαz (Hallak 1994). Yet, the expression of myristoylated, but not palmitoylated, forms of Gαo and Gαi proteins in cells led to an increased proportion of Gα proteins in the soluble cellular fraction (Mumby 1994; Galbiati 1994; Grassie 1994; Degtayarev 1994). This demonstrated that myristoylation is not critical for membrane anchoring of all subtypes of Gα proteins. It was hypothesized that myristoylation of certain Gα proteins, such as Gαi proteins, may be necessary before those Gα proteins could be palmitoylated (Galbiati 1994). This hypothesis does not hold true for all Gα proteins as some Gα proteins do not undergo myristoylation but are palmitoylated, such as Gαs and Gαq proteins (Mumby 1994). Therefore, palmitoylation may be more important than myristoylation for membrane localization of certain Gα proteins. Thus, the importance of myristoylation is not universal and is dependent on the type of Gα protein. Myristoylation can effect the ability of certain Gα proteins to stimulate downstream effectors, independent of membrane association of the Gα protein.

While non-myristoylated Gαz proteins still retained some of their ability to inhibit adenylyl cyclase activity (Wilson 1995), myristoylation of Gαi proteins is required for the inhibition of adenylyl cyclase in an in vitro, cell-free system (Taussig 1993).

Thus, while myristoylation is not required for the membrane localization of Gαi proteins, it is necessary to proper interaction between Gαi proteins and their effector enzyme. Hence, myristoylation is important for membrane localization and Gα protein-effector protein interactions for certain Gα proteins.

5.3. Palmitoylation

Because of its importance in protein localization to the membrane, palmitoylation is important for proper receptor-mediated signaling (Qanbar 2003). Palmitoylation is the covalent attachment of palmitate (16-carbon fatty acid) to any cysteine residue. No consensus sequence is required for palmitoylation and it is a reversible process (Mumby 1997). All Gα proteins, except for Gαt proteins, are palmitoylated (Wedegaertner 1995). Palmitoylation of Gα proteins is important for the localization of the Gα protein to lipid membranes (Fig. 2). In vitro assays have shown that palmitoylated Gαo subunits associate more strongly with membrane fractions than their non-palmitoylated counterparts.266 Mutations of the palmitoylation sites of Gα proteins have yielded an increased amount of mislocalized or soluble Gα proteins (Qanbar 2003; Fishburn 2000). Membrane-bound proteins or kinases may also assist in sequestering Gα proteins to the lipid membrane. Membrane-bound palmitoyltransferases (MIR16) (Zheng 2000) or Gβγγ proteins 306 K.J. DAMJANOSKA ET AL.

(Hughes 2001), acting as a temporary membrane-bound docking proteins, may recruit a non-palmitoylated Gα protein to the cell membrane. Once it is palmitoylated, the Gα protein can associate with the cell membrane directly. Other lipid modifications, such as myristoylation, or protein interactions of the Gα protein with receptors or Gβγγ proteins may localize the Gα protein after its activation.

Activation of Gαs, Gαi, and Gαq proteins by β-adrenoceptors (Mumby 1994; Degtyarev 1993), cholera toxin, or serotonin receptors (Chen 2000; Bhamre 1998) in cell culture or in tissue preparations resulted in an increase in palmitate labeling of the Gα proteins. This increase in palmitate labeling could either be due to an increase in the incorporation of palmitate into Gα proteins or rather an increase in the turnover of the palmitoylation-depalmitoylation cycle represented by the attachment of palmitate into Gα proteins. Pulse-chase methods and half-life measurements in cell culture studies have supported the latter hypothesis (Mumby 1994; Wedegaertner 1994). The exact mechanisms of how and when the Gα proteins become palmitoylated and depalmitoylated are still not known. Based on our current knowledge (Fig. 2), stimulation of receptors causes a depalmitoylation of Gα proteins, but the signal that triggers the repalmitoylation of the Gα proteins is not known. While palmitoylation is an important component to proper membrane localization, it is not clear how the activation-dependent depalmitoylation of Gα proteins fits into proper signaling. Perhaps depalmitoylation and dissociaton from the membrane are required before the Gα proteins can undergo transformation into their GTP-bound form. On the other hand, immunohistochemical evidence in transfected HEK-293 cells showed that α2A--mediated and AlF4- mediated activation of Gαq proteins did not change the cellular localization of the Gαq proteins (Hughes 2001). These Gαq proteins were still associated to the cellular membrane. This suggests that activation of Gα proteins may not necessarily lead to depalmitoylation and cellular relocation of the Gα proteins. Thus, the current model of palmitoylation/depalmitoylation of Gα proteins (Fig. 2) will most likely undergo some fine tuning in the near future. Although many of the regulatory mechanisms need to be elucidated, the regulation of palmitoylation and depalmitoylation of Gα proteins may provide us with novel pharmacological targets to control Gα protein- mediated signaling. RGS proteins, as well as G proteins, can be palmitoylated (RGS4, RGS7, RGS10, RGS16, and GAIP) (Osterhout 2003; Hollinger 2002). This lipid modification has been shown to be important for the GAP activity of RGS4, RGS10, and RGS16, and membrane association of RGS4, RGS7, and GAIP (Hollinger 2002). Thus, G protein signaling can be altered by palmitoylation of Gα proteins and/or RGS proteins. Palmitoylation of receptors also affects signaling (Qanbar 2003). Mutations on the palmitoylation sites of β2-adenoceptors (Moffett 1993; O’Dowd 1989), endothelin A (ETA) receptors (Doi 1999), m2 muscarinic receptors (Hayashi 1997), G PROTEINS 307 and somatostatin receptors (Hukovic 1998) resulted in the uncoupling of the receptors from their G proteins, thereby decreasing receptor-mediated enzyme activity. Interestingly, palmitoylation of the receptor can have differential effects on the G proteins that bind to it. The unpalmitoylated form of the human ETA receptor is less effective in stimulating Gαi and Gαq proteins but it stimulates Gαo proteins to the same degree as palmitoylated ETA receptors (Doi 1999). Site-directed mutagenesis of a palmitoyolated cysteine (C341G) on the β2-adrenergic receptors led to the receptor being highly phosphorylated and decreased receptor coupling to

Gαs proteins (Moffett 1993). Thus, post-translational lipid modifications can contribute to the specificity of a receptor for a particular G protein, its degree of interaction, and also influence other post-translational modifications.

5.4. Phosphorylation

Phosphorylation of GPCRs occurs mainly in consensus sequences in the second and third intracellular loops of these receptors. However, other intracellular portions of the receptors also can be phosphorylated. The phosphorylation of receptors can lead to the inactivation of the receptor and a reduction in signaling due to internalization and/or a disruption of the interaction between the receptor and its trimeric G protein. Phosphorylation of receptor proteins by kinases has been associated with receptor desensitization due to β--dependent or -independent internalization and inactivation of adrenergic and serotonergic receptors (Gray 2001; Lohse 1992). Phosphorylation of the gastrin-releasing peptide receptor, in the absence of can reduce receptor-mediated activation of Gα proteins by approximately 80% (Kroog 1999). This same study showed that the desensitization observed was due to a decrease in the catalysis of guanine nucleotide exchange rather than a change in the affinity of the receptor for trimeric G proteins. Hence, phosphorylation of receptors can alter intracellular signaling by internalizing the receptor, hindering receptor-Gα protein interaction, or altering guanine nucleotide exchange. Phosphorylation of G protein subunits (Chen 2001) directly can decrease the association between the trimeric G protein and receptor or between the Gα protein and effector enzyme leading to a decrease in G protein-mediated signaling.

Phosphorylation of Gαz proteins hinders them from reassociating with Gβγγ proteins (Fields 1995) and decreases the susceptibility of the Gαz protein to the GAP activity of RGSZ1 (Glick 1998). The phosphorylation of Gγ proteins, causes them to associate more with Gα proteins than with effector enzymes, thereby decreasing Gγ protein-mediated signaling.281 Phosphorylation does not necessarily have to affect G protein signaling in a negative manner. Phosphorylated forms of Gαq/11 proteins are more efficacious in stimulating phospholipase Cβ (PLCβ) activity (Liu 1996). For receptors that activate kinases, this may be one mechanism by which they can amplify their own signal by enhancing Gαq/11 protein activity (Umemori 1997). 308 K.J. DAMJANOSKA ET AL.

Phosphorylation of RGS proteins can also affect G protein signaling (Hollinger 2002). Phosphoryl-ation of RGS proteins functions to directly alter GAP activity, membrane localization, and association with 14-3-3 scaffolding proteins.186 Phosphorylation is necessary for the interaction of RGS3 and RGS7 with the scaffolding protein 14-3-3 (Benzing 2000 and 2002; Niu 2002). These same studies showed that interacting with 14-3-3 proteins decreased the ability of RGS3 proteins to interact with Gα proteins and decreased the GAP activity of RGS7 proteins (Niu 2002; Benzing 2000). After phosphorylation, GAP activity is attenuated for RGS2 (Cunningham 2001) and RGS16 (Chen 2001) proteins but is enhanced for GAIP proteins (Ogier-Denis 2000). Phosphorylation of RGS proteins can affect the activity of RGS proteins by mechanisms other than directly affecting GAP activity. For example, the RGS proteins associate with the 14-3-3 protein instead of Gα proteins, thereby increasing the duration that the Gα proteins can remain in their active form (Gα-GTP). In addition, nuclear translocation of RGS10 occurs after phosphorylation and is associated with a reduction in the ability of RGS10 to regulate Gα protein activation (Burgon 2001). Thus, phosphorylation of RGS proteins can modulate the activity of RGS, Gα, and Gβγγ proteins by a variety of mechanisms.

6. G PROTEIN-ASSOCIATED CNS DISEASES

This section will highlight psyciatric disorders associated with changes in G proteins, RGS proteins, and some GPCRs. Since each disorder will be covered in its own chapter within this book, we will only focus on changes of G proteins and related proteins or signaling cascades associated with the disease state or with its treatment. Many of the studies measure mRNA or protein levels in post-mortem tissue while others use animal models to investigate the effect of pharmacological treatments on receptor signaling and protein levels. Although we have mentioned a few studies utilizing knock-out animals, we will not provide a comprehensive overview of all the studies utilizing transgenic and knock-out animals. These genetic manipulations in whole animals occur prior to birth and, if the alteration is not fatal, may lead to complementary developmental changes, such as compensation by another protein or signaling pathway. Animals with inducible knock-out genes, where the elimination of a specific protein occurs after the animal matures, are a novel attempt to avoid the issue of developmental compensation in genetically altered animals.

6.1. Anxiety and neuroticism

Anxiety disorders encompass generalized anxiety disorder (GAD), panic disorder, social phobia, posttraumatic stress disorder, and obsessive-compulsive disorder (OCD) (Nutt 1996). Anxiety is considered to be an inappropriate fear G PROTEINS 309 response or a fear response at an inappropriate time and is associated with improper signaling from the central nucleus of the amygdala (Ninan 1999). An estimated 19 million American adults suffer from some form of anxiety (NIMH 2002). Anxiety disorders are traditionally treated with benzodiazepines, although serotonin 1A (5- HT1A) receptor agonists (buspirone) and selective serotonin reuptake inhibitors (SSRIs) are efficacious without the risks of abuse, dependence, and withdrawal effects that are associated with benzodiazepines. A better understanding of the role of the gamma amino butyric acid (GABA) system in anxiety and in the mechanism of action of benzodiazepines has led to the development of GABA receptor antagonists that target specific subunits of GABAA receptors to regulate GABA- ergic transmission. Glutamate, corticotrophin releasing factor/hormone (CRF or CRH) and substance P are other neurotransmitters that are abnormally regulated in anxiety disorders. Antagonists of CRF and substance P receptors are currently under investigation for the treatment of anxiety (Goman 2003). Subchronic treatment of rats with buspirone, a 5-HT1A receptor partial agonist, decreases Gαi1 and Gαi2 protein levels in the cerebellum (Dwivedi 1997). On the other hand, alprazolam, a benzodiazapine, and metachlorophenylpiperazine (m-

CPP), an anxiogenic drug, did not change the levels of Gαs, Gαi, or Gαq/11 proteins in a variety of brain regions. Alterations in levels of Gα proteins could be treatment- specific. Thus, the importance of Gα proteins in the etiology or treatment of anxiety is still not known. The importance of RGS2 proteins in anxiety and aggression is implicated by knockout mice (rgs-/-) displaying increased anxiety and decreased male aggression (Oliveira-dos-Santos 2000).

6.2. Autism and autistic disorders

Autism is a childhood behavioral and neurological disorder with onset prior to three years of age. The main features of autism spectrum disorders (ASD) are deficits in language, social, and emotional functioning, with significantly variable secondary symptoms of aggression, self-injurious behavior and impulsivity. Autism now affects 62-67 per 10,000 births, with boys being affected four times more frequently than girls (Bertrand 2001; Chakrabarti 2001). While the exact cause of autism is unknown, an abnormal functioning of receptors may be one mechanism involved. The theory of vaccine- related autism is actually related to the pertussis toxin found in the diphtheria, pertussis, and tetanus (DPT) vaccine. The toxin in the vaccine uncouples Gα proteins from retinoid receptors in the brain, thereby functionally uncoupling receptors from their intracellular signaling proteins (Megson 2000). A pre-existing family history of defects in Gα proteins (night blindness, , and thyroid or pituitary adenoma) increases the risk of autism due to the vaccine. 310 K.J. DAMJANOSKA ET AL.

6.3. Bipolar Disorder

Bipolar (manic-depressive) disorder is characterized by cycling episodes of depression and mania with a lifetime prevalence of 1.2% (Weissman 1988). The postmortem frontal cortex tissue of bipolar victims shows enhanced receptor-to-G protein coupling along with increased trimeric states of the G proteins (Friedman

1996). This study shows an increased expression of Gαs proteins without changes in levels of other G proteins, but all G proteins show a higher propensity to be in their trimeric state. The higher fraction of trimeric G proteins may indicate a supersensitization of G protein-mediated signaling in patients with bipolar disorder. is a common antimanic agent. In agreement with the possibility that bipolar disorder may be partially due to an increase in Gαs protein levels, bipolar patients treated with lithium showed a decrease in Gαs protein levels in the occipital cortex (Dowlatshahi 1999). Lithium treatment also decreases ADP-ribosylation of

Gαi proteins (Watanabe) and Gαi protein expression in cerebral cortex (Colin 1991) while increasing adenylyl cyclase expression in the cerebral cortex. Lithium has also been shown to decrease levels of Gαi1/2 in rat cortex and hippocampus after subchronic treatment (Dwivedi 1997). Lithium’s ability to change the levels of adenylyl cyclase, Gαi and Gαs proteins, both of which regulate adenylyl cyclase activity, indicates that bipolar disorder may be due to an altered signaling of adenylyl cyclase pathways. More recent studies suggest that lithium, valproic acid, and carbamazepine decrease the levels of inositol in the brain (Harwood 2003; Wolfson 2000; Dixon 1997; Berridge 1982). Inositol is the precursor to phosphatidyl inositol bisphosphate (PIP2) which is cleaved by activated PLC to generate (IP3) and diacylglycerol (DAG). Thus, the signaling cascade is desensitized by decreasing the amount of the required precursor (inositol) and not by modifying the function of either enzyme or G protein. The observation that lithium decreases inositol levels in critical brain areas gave rise to the most widely accepted hypothesis for bipolar disorder the inositol depletion hypothesis. One major criticism of the hypothesis is that the decrease in inositol levels does not necessarily correlate to the therapeutic onset of lithium treatment, although it has not been demonstrated that the therapeutic onset of lithium is independent of a decrease in inositol levels (Moore 1999).

6.4. Depression

Depression is an affective (mood) disorder characterized by anhedonia that affects approximately 9.5% of American adults annually (NIMH 2002). A decrease in the synaptic levels of monoamines (serotonin, norepinephrine, epinephrine) in the CNS may be an underlying cause of several mood disorders, including depression and anxiety (Delgado 200). The serotonin system has been of particular interest in antidepressant treatment after the success of selective serotonin reuptake inhibitors G PROTEINS 311

(SSRIs), such as fluoxetine (Prozac®). Although fluoxetine increases the synaptic levels of serotonin (5-HT) within a few days after administration of the drug, the symptoms of depression are not alleviated for another 2-3 weeks. This delay indicates that a mere increase in levels of synaptic monoamine is not sufficient to provide antidepressant effects. Thus, neuroadaptive changes occurring within the intracellular signaling cascade may mediate the antidepressant effects of SSRIs (Manji 1999; Li 1996; Raap 1999). In comparison to the number of studies that have examined the role of receptors in depression, there are fewer studies that have investigated changes in G proteins or RGS proteins in depression or its therapy (Donati 2003). PET scans in brains of depressed patients have generally shown decreased 5-HT1A receptor densities and increased 5-HT2A receptor densities (Dhaenen 2001). Abnormalities in the hypothalamic-pituitary-adrenal (HPA) axis are associated with depression as an attenuated release of ACTH to CRH administration is seen in depressed patients (Holsboer 2000; Heuser 1994). Clinical studies have shown that HPA activity normalizes after chronic antidepressant treatment and is associated with an improved prognosis (Inder 2001; Sonino 1996; Barden 1995). Neuroendocrine measures have shown decreased 5-HT1A receptor-mediated of ACTH and in depressed patients (Meltzer 1994 and 1995; Pitchot 1995; Lesch 1990). Treatment with chronic fluoxetine desensitizes postsynaptic 5-HT1A receptors in the hypothalamic paraventricular nucleus in rats, a phenomenon associated with decreased levels of Gαi, Gαo, and Gαz proteins (Raap 1999; Li 1997). Human studies also indicate that treatment with SSRIs induces a desensitization of hypothalamic post-synaptic 5-HT1A receptors (Lerer 1999; Berlin 1998; Sargent 1997). However, this desensitization may be a unique effect of SSRis because the monoamine oxidase inhibitor phenelzine increases levels of Gαi1/2 in the cortex and hippocampus of rats after subchronic treatment (Dwivedi 1997). The tricyclic antidepressants do not seem to affect G proteins as subchronic treatment with desipramine does not alter levels of Gαs, Gαi1/2, or Gαq/11 proteins in the brain. Hence, the effects of SSRIs on post-synaptic 5-HT1A receptors in hypothalamus and amygdala (Bosker 2001) may represent their unique therapeutic effects on a variety of other mood disorders (anxiety, eating disorders, OCD, premenstrual syndrome). On the other hand, it is likely that several different neurochemical disorders underlie the symptoms of depression. Thus, different anti-depressants can be useful to treat depression because they may alter different neurochemical abnormalities. Consistent with this possibility is the fact that approximately 30% of depressed patients do not respond to the first monotherapy with an antidepressant. Densities of β-adrenoceptors have been shown to increase, decrease, and not change in brains of depressed victims (De Paermentier 1990 and 1911). These different effects of antidepressant treatments on densities of β-adrenoceptors may be due to differences in the brain area studied, psychiatric diagnosis, and previous pharmacological treatment of the suicide victims. On the other hand, an increase in 312 K.J. DAMJANOSKA ET AL. the high-affinity state of α2A-adrenoceptors without a change in the density of α2A- adrenoceptor was observed in tissue samples of frontal cortex, hypothalamus, and locus coeruleus obtained from suicide victims with major depression (Callado 1998).

The increase in the density of the high-affinity state of α2A-adrenoceptors suggests a change in the receptor-to-G protein coupling, which may be indicative of a change that occurs within the intracellular portion of the lipid bilayer. Coincidently, increased levels of Gαs proteins have been reported in the cerebral cortex of subjects with major depressive disorder (Pacheco 1996). Hence, it is possible that the increase in the high-affinity state of α2A-adrenoceptors in individuals affected with major depression is due to an increase in the respective Gα protein, specifically Gαs protein.

6.5. Neurodegenerative diseases

Neuronal loss is the pathological trademark of neurodegenerative diseases, such as Alzheimer’s and Parkinson’s disease. Most research in neurodegeneration focuses on theories based on changes in amyloid, tau, or synuclein proteins. The amyloid and tau theories state that β-amyloid (Aβ) proteins and tau aggregate into β-amyloid plaques and neurofibrillary tangles, respectively; both being hallmarks of Alzheimer pathology. While both of these events are thought to be key in neuronal destruction, other research has shown neurotrophic growth factors (Siegel 2000), (Gandy 2003), (Puglielli 2003), and lipoproteins (Neely 2002) also to have an effect on neurodegenerative diseases. We will focus on the research that specifies the involvement of G proteins and their associated proteins.

6.5.1. Alzheimer’s disease

Alzheimer’s disease is the leading cause of dementia and affects about 4.5 million Americans (Alzheimer’s Association, Statistics 2003). Alzheimer’s disease is associated with a disruption in the coupling of Gαq proteins to their effector enzyme phospholipase C, which may lead to a reduction in glutamate, histamine, and serotonin receptor-mediated phosphoinositide hydrolysis (Cowburn 1996). This disruption occurs without changes in the levels of Gαq proteins in various brain regions of affected individuals. However, another study has shown a decrease in total levels of Gαq/11 and RGS4 proteins, without a change in the membrane levels of either protein, in parietal cortex obtained post-mortem from patients with

Alzheimer’s disease (Muma 2003). Gαs protein-mediated adenylyl cyclase activity, but not forskolin-stimulated adenylyl cyclase activity, is reduced in various brain areas from patients with Alzheimer’s disease (Cowburn 1996). Interestingly, Gαi mediated intracellular signaling does not change in brains of Alzheimer’s patients. Thus, while not all the results seem to be consistant, Alzheimer’s disease does appear to effect the signal transduction of both Gαq and Gαs proteins. G PROTEINS 313

6.5.2. Parkinson’s disease

Parkinson’s disease is characterized by abnormalities in extrapyramidal motor function. The pathology of Parkinson’s disease is the degeneration of dopaminergic neurons in the midbrain substantia nigra (Brooks 2003). These neurons project to the striatum, composed of the caudate nucleus and the putamen. It is the loss of striatal dopaminerigic innervation that leads to a decrease in the dopamine content of the striatum. While the decrease in striatal dopamine explains the abnormalities in extrapyramidal motor functions, a complete knowledge of the molecular adaptations that follow the dopaminergic denervation of the striatum is lacking. There have been a few studies that have addressed this issue. Western blot analyses show an increase in RGS9 protein levels in the caudate and putamen (or dorsal striatum) of patients with Parkinson’s disease (Tekumalla 2001). This indicates a striatal adaptation that occurs in Parkinson’s disease and may provide a target for future pharmacological agents.

6.6. Schizophrenia

Schizophrenia is a psychiatric disorder characterized by positive symptoms (visual and auditory hallucinations, delusions) and negative symptoms (apathy and lack of motivation). The original (typical) antipsychotic drugs are dopamine (D2) receptor antagonists. Atypical antipsychotic agents that are antagonists of serotonin (5-HT) receptors, with weak antagonism of D2 receptors, have been more effective than typical antipsychotic drugs in the treatment of the positive symptoms of schizophrenia and cause less extrapyramidal side-effects than typical antipsychotic drugs. An upregulation of D2 receptors (Kapur 1996) and 5-HT1A receptors (Bantick 2001) and a downregulation of 5-HT2A receptors (Hernandez 2000) have been documented in brains of schizophrenic patients post-mortem. A decrease in levels of

Gαi, Gαo, and Gαq proteins, but not of Gαs or Gβ proteins, was detected in the superior temporal cortex of schizophrenic patients (Yang 1998). Paradoxically, an upregulation of Gαi protein-coupled receptors, such as D2 and 5-HT1A receptors, was observed while a decrease in Gαi proteins was observed in another study. These data may lead us to hypothesize that D2 and 5-HT1A receptors are upregulated to compensate for a decrease in levels of Gαi proteins. In addition to changes in levels of Gα proteins, the regulation of Gα proteins might also be altered in schizophrenia. Gene microarray analysis showed a 50-84% decrease in expression of RGS4 protein in cortical areas of subjects with schizophrenia.203 RGS4 has GAP activity for both

Gαi and Gαq proteins. Since a decrease in cortical levels of Gαi and Gαq proteins was reported, a decrease in RGS4 protein levels would be one possible compensatory mechanism to counter-balance the decrease in levels of Gα proteins. These thoughts are just speculation as it is not know which proteins change first and which are 314 K.J. DAMJANOSKA ET AL. compensations. Furthermore, because of the difficulty of finding brain tissue from untreated schizophrenic patients, it is not entirely clear that the changes in the schizophrenic brain are due to the disorder or an adaptive change induced by treatment with antipsychotic drugs. Nevertheless, these studies add support to the idea that schizophrenia may be mediated by abnormal intracellular signaling due to alterations in G proteins in various brain areas.

7. CONCLUSION

The importance of G proteins and their associated signaling cascades in the etiology of neuropsychiatric disorders is receiving increasingly extensive attention. G proteins and their associated signaling proteins provide intracellular regulation and modulation of extracellular signals, which can be utilized to develop novel pharmacological agents to treat psychiatric disorders. Such medications may not have the specificity of drugs that affect specific receptors. However, medications that address post-receptor signaling proteins may be useful in disorders involving multiple receptor systems, particularly those that utilize common second messenger systems (for example, the treatment of bipolar disorder).

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